Notch signaling is triggered by extracellular receptor and ligand interactions. Notch receptors control normal cellular proliferation, differentiation, and death in multicellular organisms through a signaling cascade that is triggered by ligand-induced proteolysis. After furin-like protease cleavage at site S1, the mature Notch heterodimer is translocated into the cell membrane where it is held in an auto-inhibited state by a juxtamembrane Negative Regulatory Region (NRR) consisting of three Lin12/Notch repeats (LNR-A, B, C) and the heterodimerization (HD) domain. The HD domain is divided into N-terminal (HD1) and C-terminal (HD2) halves after cleavage at site S1. Through an uncertain mechanism, binding of ligands of the Delta/Serrate/Lag-2 (DSL) family to the extracellular EGF-repeat region relieves this inhibition and induces two additional cleavage events. First, ADAM-type metalloproteinase mediate cleavage at site S2 near the C-terminal region of the HD-2 domain, thereby releasing the extracellular domain (ECD) from the cell surface which then undergoes trans-endocytosis into the ligand-expressing cell. Next, gamma-secretase mediates cleavage at site S3 within the transmembrane domain which releases the intracellular domain of Notch (Notch-ICD) from the membrane, permitting it to translocate to the nucleus and activate the transcription of target genes (Bray, S., Nature Reviews Molecular Cell Biology, 2006, volume 7, 678-689).
The X-ray crystal structure of the human Notch2-NRR domain in an auto-inhibited conformation revealed extensive interactions between the LNR repeats and heterdimerization domains within the NRR burying the metalloprotease S2 site, suggesting that a substantial conformational movement is necessary to expose the site during activation by ligand (Gordon, W. R., et. al, Nature Structural & Molecular Biology, 2007, volume 14, 295-300). Studies suggest that stabilization of the interactions within the NRR may prevent ligand-induced Notch activation. The availability of structural information on the Notch auto-inhibited conformation provided new opportunities for the development of therapeutics, particularly antibodies that target Notch signaling. (Li, K., et. al, Journal of Biological Chemistry, 2008, volume 283, 8046-8054; Aste-Amezaga, M, et. al, PLOS ONE, 2010, volume 5, e9094; Wu, Y., et. al, Nature, 2010, volume 464, 1052-1057).
In mammalian cells, there are four known Notch receptors. Notch-1, -2, -3 and -4 have broad, overlapping patterns of expression in embryonic and adult tissues, and fulfill non-redundant roles during hematopoietic stem cell specification, T cell development, intestinal crypt cell specification and vascular development. Notch3 is expressed primarily in vascular smooth muscle cells (vSMC), various thymocyte subpopulations and the developing nervous system. Consistent with its restricted tissue distribution, targeted deletion of murine Notch3 does not lead to embryonic lethality like Notch1 and Notch2 deletion. Instead, Notch3-null mice are viable, but have defects in the maturation and differentiation of vSMCs (Domenga, V., et. al, Genes and Development, 2004, volume 18, 2730-2735).
Notch activation is oncogenic in many contexts; constitutively active, intracellular forms of all four Notch homologues function as oncogenes in vitro and in transgenic mouse models. Recent studies indicate that Notch3 is often amplified and overexpressed in various human solid tumors and the over-expression of developmental signaling pathways, such as Notch3, in human cancers implicates them as key mediators of tumorigenesis. Several strategies are in development to block Notch signaling for therapeutic purposes in cancer; however there is still a need in the art for more potent and efficacious anti-Notch targeted therapies for the treatment of cancer.
Antibody-drug conjugates (ADCs) combine the specificity and targeting of high affinity antibodies with the cytotoxicity of a therapeutic agent, such as cytotoxic agents, biological response modifiers, enzymes, apoptosis-inducing agents, and radioisotopes. Release of therapeutic agents from the antibody can require trafficking and localization of the antibody-drug conjugate to lysosomes and both Notch3-ECD and Notch3-ICD undergo lysosomal degradation, thus antibodies that bind Notch3 are expected to traffic to the lysosome (Jia L, et. al, International Journal of Biochemistry and Cell Biology, 2009, volume 41, 2594-2598). The present invention provides novel anti-Notch3 antibodies and antibody-drug conjugates that fulfill an unmet clinical need in the diagnosis and therapeutic use in the treatment of cancer.